Understanding the molecular events mediating HIV binding and entry into susceptible cells is critical to the development of effective AIDS therapeutics and vaccines. In FY 1995, we investigated the mechanisms used by monocytotropic/primary (MT) and T cell line (T) tropic virus to gain entry into CD4+ cells. We continued our studies on the role of cell surface heparan sulfate (HS) proteoglycans in mediating virus attachment and infection and found that different entry requirements exist for MT and T tropic viruses. T tropic viruses are dependent on HS for binding and infection, while MT viruses show HS independence and a marked resistance to blocking by sulfated polyanions. Using envelope chimeric constructs, we mapped HS dependence specifically to the V3 region of gp120. Our efforts to identify virus entry cofactors revealed that a cell surface protease, CD26, plays a role in the infection of MT but not T tropic viruses. Using cell clones selected for differential CD26 expression, we found a correlation between the levels of expression of the ectopeptidase CD26 and the entry, replication and cytopathicity of MT/primary viruses. This important finding helps to resolve some the controversy surrounding the role of this ectopeptidase in HIV infection and may explain why CD26 positive cells are lost from the circulation in HIV infected patients. Several of these unique cell clones generated during the course of the entry studies are proving useful for isolation and analysis of primary patient isolates. These systems should provide important tools to identify cell surface molecules mediating virus infection and have applications for drug screening, analysis of patient isolates to allow individualization of HIV drug therapy, and for evaluating humoral and cellular responses to prophylactic and therapeutic vaccines. Roderiquez, G., Oravecz, T., Yanagashita, M., Bou-Habib, D., Mostowski, H., and Norcross, M. Mediation of HIV-1 binding by interacton of cell surface heparan sulfate proteoglycans with the V3 region of envelope gp120-gp41. J. Virology 69, 2233-2239 (1995) Oravecz, T., Roderiquez, G., Koffi, J., Wang, J., Ditto, M., Bou-Habib, D.C., Lusso, P., and Norcross, M.A.. CD26 expression correlates with entry, replication, and cytopathicity of monocytotropic HIV-1 strains in a T-cell line. Nature Medicine 9, 919-926 (1995)